The use of colloidal particles and magnetic particles to bind a compound has long been known and used in industrial and laboratory procedures. For example, crosslinked polystyrene-divinylbenzene beads, among the earliest and most widely used particles, have been used in organic synthesis, catalysis and the biotechnical arts, especially immunology. In combination with the appropriate reagents, the particles have been used to remove specific cells from a sample containing a plurality of cell types or to enhance the results of instrumental biomedical assays. For example, polystyrene microbeads have been used as a standard in place of natural cells in order to reduce test-to-test variances. Unless specified otherwise, the terms "particles", "spheroids", "spheres", "microspheres" and "beads" as used herein, are interchangeable.
In the 1970s, there emerged a phenomena known as SERS or Surface-Enhanced Raman Scattering [9,10]. Using thin metal films or coatings deposited on a substrate, it was found that small structures of conducting metals such as gold or silver showed unusual light scattering and absorption effects; provided that the structures had dimensions about that or smaller than the wavelength of the exciting radiation being used. These effects were found to be due to the unique refractive index versus wavelength properties of such structures. Various methods have been developed for depositing metal coatings on various substrates. However, achieving a uniform metal coating has been difficult, especially on three-dimensional substrates like polymeric or glass beads.
The preparation of gold- and silver-coated substrates, including spheroidal particles, has been described in numerous publications. In the early stages of this art, Teflon.RTM. spheres on a flat glass surface were coated with silver structures or "bumps" of 100-200 nm (nanometers) diameter and 75-80 nm thickness [1]. The silver metal was deposited using well known vapor deposition techniques. Raman spectroscopy of organic substances adsorbed on the silver surfaces showed that there was enhanced Raman scattering relative to standard techniques. Metal vapor deposition has also been the standard method of applying thin gold coatings onto samples prepared for scanning electron microscopy (SEM) [2]. In both these instances, the particles were not entirely coated with the selected metal because the particulate samples were supported by some substrate. For example, the particles were scattered on a glass slide before vapor deposition of the metal. The portion or area of the particle in contact with the substrate was thus shielded from the coating vapor. The vapor deposition technique might better be used with aerosols, provided that the particles prepared would be large enough to scatter light and would remain suspended after coating with a heavy metal. Regardless, one can conclude that the vapor deposition technique would not be representative of metal coated spheres prepared in situ in solution and transferred into a liquid system to yield a colloidal suspension of uniformly coated particles.
Heterocoagulation methods using polymer spheres and based on established procedures [3,4] for the formation of gold or silver hydrosols have been studied by the present inventors. When using amidine or sulfate polystyrene particles or latices as the substrate in an aqueous medium, no detectable amounts of metal were deposited on the substrate. Instead, using gold hydrosols and polystyrene particles, as an example, separate colloidal gold metal particles of about 20 nm diameter were formed in the aqueous medium. Thus, there existed separate and distinct gold and polystyrene particles. Extinction spectra indicated that not even small gold bumps, analogous to the silver bumps or of smaller size, were deposited on the polystyrene particles. The technical literature, however, contains some reports of the deposition of certain metal compounds on polymer microspheres using heterocoagulation techniques [5,6; yttrium, aluminum, zirconium and chromium were reported deposited]. S. Margel reported the deposition of transition metals, including gold and silver, on polyaldehyde microspheres [8]. An aqueous suspension of microspheres in a metal ion containing solution, followed by metal ion reduction using a reducing agent such as sodium borohydride, was used to prepare the metal coated microspheres.
In the biotechnical arts, interest in metal-coated colloidal particles or substrates continues because, among other applications, these particles have the ability to enhance the right angle light scatter in flow cytometric forward versus side scatter histograms. It is expected that a thin coating of gold or silver on relatively large polystyrene particles (2.15 .mu.m; 2150 nm) will show behavior similar to metal islands deposited on flat surfaces or microlithographically etched arrays and on roughened metal electrode surfaces. Each of these enhances light scattering with excitation in the visible light region into the characteristic plasmon or collective free electron oscillation bands of these metal structures.
In elastic scattering of light from small particles, the extinction is dependent on (1) the size of the particle relative to the wavelength of light being used; that is, the ratio 2.pi.a/.lambda.; and (2) the refractive index of the irradiated material. Gustav Mie, in an effort to explain the colors of colloidal gold suspensions, was the first to obtain a general solution for the scattering of light by a homogeneous sphere [11-13]. The extinction spectrum of small particles has two components- absorption and scattering. The extinction, absorption and scattering efficiencies of small metallic spheres of copper, silver and gold have been calculated using Lorenz-Mie scattering theory [14-15]. The extinction band of colloidal gold spheres of 20 nm diameter in water has a maximum at 520 nm. Similar silver spheres show a maximum at 400 nm. Calculations have shown that at particular excitation wavelengths in the blue and red regions of the visible spectrum of gold and silver spheres of selected radii, scattering will dominate the extinction and absorption will be very small. For example, calculations involving silver spheres predict that for a small particle of about 5 nm radius, absorption dominates the extinction. For particles of about 50 and 500 nm radius, scattering dominates the extinction and absorption is of low intensity. In reality, since the deposited structures of gold and silver are not the isolated metal spheres of theory, the predicted wavelength dependencies of the efficiency factors will have to be modified to account for the various undefinable shapes of the individually deposited metallic bumps and for interactions between neighboring bumps. Both of these reality effects tend to broaden the extinction band and shift its maximum to longer wavelengths. In addition, the refractive index of actual gold and silver structures may not be the same as the bulk metal values that are used in the calculations.
Experiments with gold hydrosols show that elastic scattering is enhanced by excitation into the small particle plasmon resonances which were observed near 700 nm for small gold particles [16]. For microstructural gold or silver bumps on polystyrene microspheres, the bumps being about 50-200 nm in size and having about 50-200 nm spacing between themselves, elastic light scattering can be use to distinguish polystyrene particles, gold-coated polystyrene particles and silver-coated polystyrene particles from each other. The distinctions are made in forward versus side or orthogonal scatter histograms such as those obtained using a flow cytometer [17]. In fact, attempts have been made to use 40 nm diameter gold colloids with selected antibodies as light scattering probes in flow cytometry [18-20]. The results showed insufficient or less than optimal resolution of immunogold-labelled cells. The gold label increased side scatter signal amplitudes more than tenfold when 632.8 nm He-Ne excitation was used. Bohmer and King [18]proposed that gold particles larger than 40 nm might be even more useful in obtaining ever stronger light scatter signals. They conceded, however, that no such particle-antibody conjugates were available. As noted, the calculations of Messinger et al., Ref. 14, for gold spheres of 22 nm radius showed that absorption is still slightly greater than scattering at 632.8 nm.
The preparation in liquid medium of gold particles of uniform size, spherical shape and larger than 40 nm diameter is very difficult to accomplish unless gravitational effects on the heavy metal are absent. This was shown in the experiments of Frens [21] who used the citrate method to prepare six gold suspensions of different particle size ranging from 16 to 147 nm diameter. Gold particles greater than 40 nm in diameter showed an increased tendency to coagulate in the presence of electrolytes and to form polydispersed, nonspherical particles. In contrast to these metallic particles, buoyant polystyrene latex particles of low density, i.e. 1.05 g/cc versus 18.88 g/cc for gold and 10.5 g/cc for silver, can be obtained readily. These polystyrene particles can have a variety of surface functional groups, are available in various sizes and possess exceptional uniformity in their size and spherical shape. This uniformity in size and shape will be preserved when the particles are coated with a thin layer of gold or silver. For example, polystyrene particles of 2.15, 1.59, 1.01, 0.604 and 0.294 .mu.m diameter can be coated with a thin layer of gold or silver, the thickness of the layer ranging from greater-than-zero to about 200 nm, depending on deposition conditions. Since the light scattering properties of colloidal particles depend only on the chemical composition and structure of the outermost layer of the particles, gold- or silver-coated polystyrene particles will have the same light scattering properties as finely dispersed pure gold or silver particles of the same size. The metal-coated polystyrene particles, however, are easier to keep suspended in solution because the density of such particles is considerably less that of pure metal particles of the same size. Gold or silver particles of, for example, 2.15 .mu.M diameter would be very difficult to keep suspended. Lastly, there is a considerable cost saving because less precious metal is used in preparing metal-coated polystyrene particles of a given diameter than would be used in preparing pure precious metal particles of the same size.
Gold- or silver-coated particles 40 nm or smaller [1 nm=10.sup.-9 meter (m)] in diameter cannot be seen using an ordinary light microscope. Visualization of submicron particles, those less than 10.sup.-6 m, requires the use of a special method whereby the particles are viewed using bright field or epi-polarization microscopy and electronically enhancing the contrast of the resulting image [22]. Larger particles, such as those in the 0.3-2.5 .mu.m range, which have a thin gold or silver coating can be visualized easily in an ordinary light microscope. Larger gold-coated particles are blue-purple in color and larger silver-coated particles are dark green-grey or black.
By themselves, uniform polystyrene particles of diameter 0.3-2.5 .mu.M show extensive light scattering because their large size gives 2.pi.a/.lambda. values greater than unity for light wavelengths in the visible region. The uniform size and shape of these particles makes them an ideal support for studying the effects of small gold or silver structures on various light scatter phenomena. Under favorable excitation wavelength conditions, at least one of the assorted metal coated polystyrene microspheres can be made to produce its maximum light scatter. The intensity of the light scatter will depend mainly on the refractive index properties of the small gold or silver structures present on the microspheres. These metallic structures also will give a degree of granularity or shape complexity to the entire particle which is similar to the granularity or shape complexity seen in biological cells [23]. The magnitude of this granularity or the scale of surface roughness (complexity) will mainly affect the amplitude of the side scatter. Forward scatter, also related to the size of the composite particle [24], is altered very little by size considerations alone when a thin metal coating, bump size of 200 nm or less, is applied to, for example, a 2150 nm (2.15 .mu.m) diameter polystyrene particle. However, extinction properties of the metal coating must be taken into account when considering the amplitude of the forward scatter obtained using the metal coated polystyrene beads.
In the biotechnical arts, surface plasmon resonance has been used as a probe of the chemical environment near the surface of a thin film of metal evaporated onto a glass slide. Ivar Giaever reported using an island of indium or indium-gold as the substrate to adsorb a protein [25, 26]. A darkening of the slide was visually observed when an antigenic species present on the metallic surface was allowed to react with its corresponding antibody. This darkening phenomena was explained on the basis of the refractive index properties of the small indium particles [27]. At the wavelength of the incident light used in the experiment, 300 nm, Bohren and Huffman [27] showed that absorption, not scattering as proposed by Giaever, dominated the extinction and led to a a darkening of immunological slides when they were observed by transmitted light. Pharmacia Biosensor AB (Piscataway, N.J.) markets a similar surface plasmon sensor. The sensor consists of a thin gold film on the surface of the glass slide. The Pharmacia sensor is used with Pharmacia's BIAcore.RTM. instrument to monitor protein-protein and protein-ligand interactions by measuring binding and dissociation events as they occur [28].
One object of the present invention is to prepare uniform colloidal particles coated with a thin metallic layer, preferably colloidal particles which have a thin, uniform peripheral coating of metallic gold or silver. Another object of the invention is to use such metal-coated colloidal particles as shifting agents in various instrumental methods, including flow cytometry and Raman spectroscopy. A method is taught for preparing the particles of the invention and data is presented illustrating the utility of the invention.